TUBEROUS SCLEROSIS 2 GENE AND USES THEREOF

Abstract: Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by widespread development of growths in many tissues and organs. A gene (TSC2) is identified on chromosome 16 which is mutated in TSC and which may behave as a tumour suppressor. Screening of actual or suspected TSC patients for normal or mutated TSC2 can be used for diagnostic purposes. TSC2 protein (tuberin) may be used to treat or prevent unrestrained cell division and/or tumour development in patients with or without TSC.

Description: T Π-RKROUP SCLEROSIS 2 GENE AND USES THEREOF The present invention relates to the tuberous sclerosis 2 (TSC2) gene, mutations thereof in patients having TSC2-associated disorders, the protein encoded by the TSC2 gene, and their uses in diagnosis and therapy. Background to the Invention All references mentioned hereinbelow are listed at the end of this description and are herein incorporated by reference in their entirety. Tuberous sclerosis (TSC) is an autosomal dominant disorder, classified as a phakomatosis (van der Hoeve, 1933) and characterised by the widespread development of growths, usually described as hamartomata, in many tissues and organs. The unpredictable distribution of these lesions, particularly within the brain, eyes, skin, kidneys, heart, lungs and skeleton results in a wide variety of signs, symptoms and complications (Gomez, 1988). Although most frequently diagnosed as a result of neurological or derma tologica 1 manifestations, renal disease was found to be the leading cause of mortality (11/40 deaths) in the largest series of TSC deaths reported "so far. Renal complications including haemorrhage, hypertension and end stage renal disease (ESRD) are associated with the development of cysts and hamartomatous growths (angiomyolipomata) in the kidneys. Angiomyolipomata probably arise due to coexistent inactivating constitutional and somatic mutations, consistent with the TSC genes functioning as tumour or growt h suppressor genes (Green et al (1994) and Green et al (in press)). Therefore the frequency of diagnosed cases is likely to under-represent true prevalence which may be as high as 1 in 5,800 (Osborne et al., 1991). T h e pathogenesis of TSC is poorly understood and efforts . to establish the primary underlying defect have focused on positional cloning of the causative gene(s). Linkage studies have established locus heterogeneity (Sampson et al., 1989a & 1992, Haines et al., 1991a&b, Janssen et al. , 1991, Povey et al.,1991, Northrup et al., 1992) with disease determining genes on chromosomes 9 (Fryer et al., 1987) and 16 (Kandt et al., 1992) leading to apparently indistinguishable phenotypes. In most, if not all, affected multigeneration families the disease can be accounted for by the gene at one or other of these loci (Kwiatkowski et al . , 1993) . The Genome Database Nomenclature Committee recently agreed that the loci on chromosomes 9 and 16 should be termed TSC1 and TSC2 respectively. Analysis of meiotic recombination events in TSC families has refined the positions of TSC1 and TSC2 to small regions in the telomeric chromosomal bands 9q34.3 and 16pl3.3. The candidate region at 16pl3.3 extends between the markers MS205.2 (D16S309) and 16AC2.5 (D16S291) (Kwiatkowski et al., 1993), representing an estimated 1.5 megabases of DNA. Loss of heterozygosity for alleles at 16p has been observed in hamartomata from TSC patients (Green and Yates, 1993; Smith et al . , 1993), indicating that a second somatic mutation may be required to produce the TSC phenotype at a cellular level. This observation is consistent with the chromosome 16 TSC gene acting as a tumour suppressor, a feature shared by genes causing the other phakomatoses, neurofibromatosis type 1 (NF1) (Legius et al., 1993) and type 2 (NF2) (Trofatter et al., 1993), and von Hippel-Lindau disease (VHL) (Latif et al., 1993). If a two-hit mechanism, as proposed by Knudson (1971), does apply to TSC, then inactivating constitutional mutations would be anticipated. TSC has not been noted in individuals with the chromosome 16 α- thalassaemia/mental retardation syndrome (ATR-16) and terminal deletions of 16p which extend into the distal part of the candidate region ( ilkie et al., 1990). The inventors of the present invention therefore investigated the proximal part of the candidate region for deletions. Some 60% of TSC cases appear to represent new mutations (Sampson et al., 1989b) and the inventors reasoned that a proportion of these might be large deletions. Such deletions, detectable by pulsed field gel electrophoresis (PFGE), would greatly facilitate identification of the gene, as has been demonstrated in NF1, NF2 and VHL (Viskochil et al., 1990; Trofatter et al., 1993; Latif et al. , 1993). The inventors have now identified 5 TSC associated constitutional interstitial deletions of between 30 and 75 kb in the proximal part of the candidate region. These have been mapped to a 120kb segment from which the inventors have identified a number of genes, one of which was disrupted by all the deletions. Mutation analysis and expression studies provide strong evidence that this gene, which we term TSC2, is the chromosome 16 tuberous sclerosis determining gene. Summary of the Invention Accordingly, in one aspect this invention provides an isolated, purified or recombinant nucleic acid sequence comprising: - (a) a TSC2 gene or its complementary strand, (b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above, (e) a fragment of a molecule defined in (a) or (b) above. In particular, there is provided a DNA molecule having a sequence corresponding to all or a portion of the nucleotide sequence of Figure 3, or a complementary sequence, or a sequence which hybridises to any of the above sequences. In another aspect this invention provides a purified DNA molecule characterised as follows :- (i) it is present in the telomeric chromosomal band 16p 13.3, (ii) it is mutated in TSC patients, (iii) it lies between markers GGG1 and 16AC2.. The sequence is preferably contained in cosmids ZDS-5 and LADS-4. The DNA may be genomic but it is preferred for it to be a cDNA. The TSC2 gene described herein is a gene found on human chromosone 16, and the results of familial studies described herein form the basis for concluding that this TSC2 gene encodes a protein called TSC2 protein or tuberin which has a role in the prevention or suppression of TSC. The TSC2 gene therefore includes the DNA sequences shown in Figure 3, and all functional equivalents . The gene furthermore includes regulatory regions which control the expression of the TSC2 coding sequence, including promotor, enhancer and terminator regions. Other DNA sequences such as introns spliced from the end-product TSC2 RNA transcript are also encompassed. Although work has been carried out in relation to the human gene, the corresponding genetic and functional sequences present in lower animals are also encompassed. The present invention therefore further provides a TSC2 gene or its complementary strand having the sequence according to Figure 3. In particular, it provides a TSC2 gene or its complementary strand having the sequence of Figure 3 which gene or strand is mutated in some TSC patients (more specifically, TSC2 patients) . The invention further provides a nucleic acid sequence comprising a mutant TSC2 gene, especially one selected from a sequence comprising a sequence according to Figure 3 when: (a) [WS-13] about 32kb are deleted flanked by CW13 and CW9; (b) [ S-9] about 46kb are deleted with breakpoints in SM9 and C 12; (c) [ S-211] about 75kb are deleted with breakpoints between CW9 and C 15 distally, and between CW23 and CW21 proximally; (d) [WS-97] about 75kb are deleted between BFS2 and SM9 distally, and within CW20 proximally; (e) [ S-53] about 35kb are deleted between, distally, C 23 next to JHl and, proximally, such that O.δkb of TSC2 is deleted, the deletion lying proximally between SH6 and JH13; (f) [WS212] about 75kb are deleted between SM9-CW9 distally and the TSC2 3'UTR proximally as shown in Figure 8; (g) [WS-215] about lδOkb are deleted between CW20 and C 10-C 36 as shown in Figure 8; (h) [WS-227] about 50kb are deleted between C 20 and JH11 as shown in Figure 8; (i) [WS-219] about 27kb are deleted between JHl and JH6 as shown in Figure 8; and (j) [ S-250] about lδOkb are deleted in CW20 as shown in Figure 8. This invention also extends to a purified RNA molecule having a sequence corresponding to all or a portion of the nucleotide sequence of Figure 3, or a complementary sequence, or a sequence which hybridises to any of the above sequences. In another aspect, the invention provides a nucleic acid probe having a sequence as set out above; in particular, this invention extends to a purified nucleic acid probe which hybridises to at least a portion of the DNA or RNA molecule of any of the preceding claims. Preferably, the probe includes a label such as a radiolabel, for example a * ^P label. In another aspect, this invention provides a purified DNA or RNA coding for a protein comprising the amino acid sequence of Figure 3, or a protein polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein. The DNA molecule defined above may be incorporated in a recombinant cloning vector for expressing a protein having the amino acid sequence of Figure 3, or a protein or a polypeptide having at least one functional domain or active site in common with said protein. In another aspect, the invention provides a polypeptide encoded by a sequence as set out above, or having the amino acid sequence according to the amino acid sequence of Figure 3, or a protein or polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein. In particular, there is provided an isolated, purified or recombinant polypeptide comprising a TSC2 protein or a mutant or variant thereof or encoded by a sequence set out above or a variant thereof having substantially the same activity as the TSC2 protein. This invention also provides an in vitro method of determining whether an individual is likely to be affected with tuberous sclerosis, comprising the steps of assaying a sample from the individual to determine the presence and/or amount of TSC2 protein or polypeptide having the amino acid sequence of Figure 3. Additionally or alternatively, a sample may be assayed to determine the presence and/or amount of mRNA coding for the protein or polypeptide having the amino acid sequence of Figure 3, or to determine the fragment lengths of fragments of nucleotide sequences coding for the protein or polypeptide of Figure 3, or to detect inactivating mutations in DNA coding for a protein having the amino acid sequence of Figure 3 or a protein having homologous properties. A method according to the present invention may comprise detecting a TSC2-associated disorder in a patient suspected of having or having predisposition to, said disorder, the method comprising detecting the presence of and/or evaluating the characteristics of TSC2 DNA, TSC2 mRNA and/or TSC2 protein in a sample taken from the patient. Such method may comprise detecting and/or evaluating whether the TSC2 DNA is deleted, missing, mutated, aberrant or not expressing normal TSC2 protein. One way of carrying out such a method comprises : A. taking a biological, tissue or biopsy sample from the patient; B. detecting the presence of and/or evaluating the characteristics of TSC2 DNA, TSC2 mRNA and/or TSC2 protein in the sample to obtain a first set of results; C. comparing the first set of results with a second set of results obtained using the same or similar methodology for an individual not suspected of having said disorders; and if the first and second sets of results differ in that the TSC2 DNA is deleted, missing, aberrant, mutated or not expressing TSC2 protein then that indicates the presence, predisposition or tendency of the patient to develop said disorders. A specif ic method according to the invention comprises extracting a sample of TSC2 DNA or DNA from the TSC2 l o cu s purport ing to be TS C 2 DNA f rom a patient , cultivating the sample iii vitro and analysing the resulting protein, and comparing the resulting protein with normal TSC2 protein according to the well- established Protein Truncation Test. Less sensitive tests include analysis of RNA using RT PCR (reverse trans criptas e polymerase chain 5 reaction) and examination of genomic DNA. On the other hand, if step C of the method is replaced by: C. comparing the first set of results with a second set of results obtained using the same or 10 similar methodology in an individual known to have the or at least one of said disorder(s); and if the first and second sets of results are substantially identical, this indicates that the TSC2 DNA in the patient is deleted, mutated or not expressing normal TSC2 protein. 15. The invention further provides a method of characterising a mutation in a subject suspected of having a mutation in the TSC2 gene, which method comprises : A. amplifying each of the exons in the TSC2 20 gene of the subject; B. denaturing the complementary strands of the amplified exons ; C. diluting the denatured separate, complementary strands to allow each single-stranded DNA 25 molecule to assume a secondary structural conformation; D. subjecting the DNA molecule to electrophoresis under non-denaturing conditions; E. comparing the electrophoresis pattern of the single-stranded molecule with the electrophoresis 30 pattern of a single-stranded molecule containing the same amplified exon from a control individual which has either a normal or TSC2 heterozygous genotype; and F. sequencing any amplification product which has an electrophoretic pattern different from the 35 pattern obtained from the DNA of the control individual . The invention also extends to a diagnostic kit for carrying out a method as set out above, comprising nucleic acid primers for amplifying a fragment of the DNA or RNA sequences defined above. Another embodiment of kit may combine one or more substances for digesting a sample to provide EcoRI fragments and a DNA probe as previously defined. Still further, a kit may include a nucleic acid probe capable of hybridising to the DNA or RNA molecule previously defined. The protein (tuberin) described herein may be used to treat patients affected by or likely to be affected by TSC2-associated disorder such as tuberous sclerosis (TSC) . Thus the protein or a polypeptide or hybrid protein having a homologous function to the protein may be administered to a patient to alleviate or avoid the effects of a TSC2-associated disorder. As described hereinbelow, it is believed that TSC2 and the tuberin protein are involved in preventing the development of cancers in patients with or without TSC. Accordingly, the present invention further provides a method for suppressing tumour development or preventing unrestrained cell division or treating a TSC2- associated tumour which method comprises providing a functional TSC2 gene to the desired cells of the patient such as to allow expression of tuberin therein or by providing tuberin or a tuberin functional mimic (e.g. a hybrid protein having a homologous function) therein, in an amount sufficient to have the desired effect such as a cell growth regulating or tumour suppressing effect. The invention extends to any inventive combination of the features described above or in the following description. Brief Description of the Drawings:- Figure 1 is a physical map of chromosome 16 pter; Figure 2 i s a detail ed map of the TSC area of chromosome 16 ; Figure 3 is the nucleotide sequence ( cDNA) of TSC2 gene and its predicted protein also shown in ( SEQ ID No : 1 : ) ; Figure 4 shows the homology between the predicted protein sequence of tuberin (amino acids 1593-1631) and amino-terminal domains of Human GAP3 and Murine GAP; and Figure 5 is a restriction map to show the genomic distribution of TSC2. Figure 6 is the result of PFGE analysis of deletions in TSC individuals. Figures 7a-c are the results of analysis of the five small deletions affecting TSC2 represented in the restriction map of Figure 5. Figure 8 is a map of the TSC2 and PKD1 region of chromosome 16. Figure 1 is a map of the terminal region of chromosome 16p and shows the TSC2 candidate region determined by linkage analysis between MS205.2 (D16S309) and 16AC2.5 (D16S291) . The size of the terminal deletion found in ATR-16 patient BO is shown (top) (Summarised from Harris et al., 1990; Harris et al., 1991; Ger ino et al., 1992; Rack et al., 1993; Wilkie et al. , 1990; Germino et al. , 1993 and Kwiatkowski et al. , 1993). Expanded below is a detailed map of the proximal TSC2 candidate region showing the Clal (C) sites, the breakpoints of the two somatic cell hybrids N-OHl and P-M H2A and the positions of existing and selected new DNA probes. The positions of cosmids within the contig are shown below the map: 1, JCI; 2, JCII; 3, CC1; 4, CC1-2; 5, CBFS1; 6, C 9D; 7, LADS4; 8, C 12I; 9, JH1K; 10, ZDS5; 11, SMII. In Figure 2, a detailed map of the TSC area of chromosome 16, genomic sites for the enzymes BssHII, B; Mlul, M; NotI, N; Nrul, R; SacII, S and a partial map of EcoRI (E) sites are shown. The open boxes indicate the size and location of genomic probes (see Experimental Procedures for details). The solid boxes show the sizes of transcripts and their orientations on the chromosome are marked with arrows. The genomic extent of each gene is indicated with brackets. The full proximal extent of 3A3 is unknown. cDNA clones comprising the TSC2 gene are shown enlarged below. The size and location of TSC-associated deletions are shown above the map with dashed lines indicating regions of uncertainty. The S-13 deletion' is 32 kb and flanked by C 13 and C 9. A 7 kb EcoRI breakpoint fragment is seen with these two probes (Figure 3c) . WS-9 is a 46 kb deletion with the breakpoints in SM9 and CW12. An 8kb EcoRI breakpoint fragment is seen with these probes. The S-211 deletion is " 75 kb and the breakpoints lie between C 9 and CW15 distally, and between C 23 and C 21. The distal breakpoint of S-97 is between BFS2 and SM9 and proximally within C 20, with a region of approximately 75kb deleted. The S-53 deletion is ~35kb and the distal breakpoint lies within C 23, proximal to JHl. The proximal 0.6kb of TSC2 is deleted. The exact location of the proximal breakpoint of WS-53 is unknown. In Figure 3, the predicted protein is shown below the DNA sequence (also shown in SEQ ID No: 2: ), assuming that translation begins at the first in-frame methionine of the long open reading frame. The cross-hatched grey bar denotes the GAP3 related domain (amino acids 1593- 1631) . The double underlining marks the possible membrane spanning regions. The dotted line indicates a potential leucine zipper starting at amino acids 81. .r_ r indicates the repeated motif H A V E/L A L W/L K A at amino acid 76-84 and 99-107. Possible N-linked glycosylation sites (@) are marked at amino acids 1037, 1205, 1499, and 1628. Serine (S) and threonine (T) residues that are potentially phosphorylated by cAMP- and cGMP- dependent protein kinases (upward arrowheads) (Glass et al. , 1986) , protein kinase C (right arrowheads) (Woodgett et al.,1986), or casein kinase 2 (downward arrowheads) (Pinna, 1990), and possible tyrosine (Y) kinase phosphorylation sites (#) (Patschinsky et al., 1982) are indicated. Two potential polyadenylation signals at bases 5425 and 5429 (underlined) and polyadenylation cleavage sites are indicated ("). Cleavage occurs immediately before or after the marked base. In Figure 4, identical amino acids are boxed. Asterisks indicate identical, or interchangeable amino acids, which are shared between tuberin and at least one of the GAP proteins. Interchangeable amino acids were identified using the criteria of Dayhoff et al (1978). The GenBank Accession number for the Homo Sapiens tuberin mRNA sequence is X75621. In Figure 5, Genomic probes (CW26, CW12, CW18) and cDNA probes (E0.5, El.6, E0.7, E2.5) are represented by solid bars, and the position of 5 small deletions (hatched bars) affecting the gene. Exonic EcoRI sites and the 5 ' and 3 ' ends of the gene are linked to the genomic map by the diagonal lines. Figure 6 shows: (a) PFGE of Mlul-digested DNA from TSC patients and controls probed with the clones CW21 (WS-9. WS-13. WS- 97) and JHl (WS-53), which detect an ~120kb fragment in normal individuals (N) and additional smaller fragments in the patients. CW21 is deleted in patient WS-53 and so does not recognise the aberrant ~90kb fragment. The WS-97 deletion removes ~75kb including the distal Mlul site, producing an ~74kb junction fragment (see Figure 2). (b) PFGE of Nrul-digested DNA of a normal control (N) and WS-211 (211) hybridized with probes flanking the breakpoint at the distal end (CW9 and CW15) and at the proximal end (CW23 and CW21) of the deletion. As well as the normal ~150kb fragment, the same ~80kb breakpoint fragment (shown by an arrow) is seen, with the two markers outside of the deletion (CW9 and CW21). CW15 is completely deleted (no breakpoint fragment), while CW23 is mostly deleted, although a faint ~80kb fragment can be seen in the WS-211 track. (c) EcoRI-digested DNA of normal control (N) and WS-13 (13) separated on a conventional gel and hybridised with probes (CW9 and CW13) which flank the deletion (see Figure 2) . The same 7kb breakpoint fragment (shown by arrows) is seen with both markers, consistent with a deletion of 32kb, ending within the EcoRI fragments seen by these probes. Figure 7 shows: (a) Southern blot analysis in cases 5773 and 1737. Hindi11- and BamHI-digested DNAs from the patients (P) and an unrelated control (N) were hybridised with cDNA probe E 0.7. This probe detects adjacent Hindlll fragments of ~14kb and 2.5kb and a single BamHI fragment of " 14kb. In case 5773, a deletion of " lkb within the BamHI fragment removes a HindiII site to produce a junction fragment of ~16kb. The ~4kb deletion in case 1737 produces novel Hindlll and BamHI fragments of ~10kb. Adjacent fragments were normal. (b) Southern blot analysis of the de novo deletion in case WS-11. EcoRI-digested DNA from the patient (11), father (F) and mother (M) was hybridised with probes E 0.7, CW12, El.6 and CW18. E0.7 detects the normal 18kb fragment in WS-11 and both parents and an additional 17kb fragment in WS-11 alone. CW12 detects the normal 4kb fragment in WS-11 and both parents and the additional 17kb fragment in WS-11 alone, demonstrating that the 17kb fragment El.6 spans the EcoRI site that is deleted in formation of the junction fragment and so detects both normal fragments of 4kb and 18kb and the 17kb junction fragment. CW18 is deleted on the mutant chromosome and so fails to detect the junction fragment. A Hindlll junction fragment and novel small BamHI fragment were also seen by Southern analysis, and probes recognising a variable number of tandem repeat polymorphisms were used to confirm biological parentage (data not shown) . (c) The TSC2 cDNA clone 2A6 hybridised to a Northern blot containing 1 g of lymphocyte mRNA from a normal control (N) and TSC patient WS-11 (11), who has an intragenic genomic deletion (see [b] ) . An additional transcript (shown by an arrowhead) "lkb smaller than normal is seen in WS-11. Figure 8 shows genomic sites for the enzymes Mlul (M), Clal (C), Pvul (P) and Nrul (R) are shown. Positions of single copy probes and cosmids used to screen for deletions are shown below the line which represents " 400 kb of genomic DNA. The genomic distribution of the approximately 45 kb TSC2 gene and known extent of the PKD1 gene are indicated above. The hatched area represents an " 50 kb region which is duplicated more proximally on chromosome 16p. Detailed Description of the Drawings Deletions in The TSC Candidate Region An ATR-16 patient (BO), with a constitutional deletion at 16p (Wilkie et al . , 1990) which extends into the TSC candidate region (Figure 1) , was specifically reassessed for signs of TSC (by clinical evaluation, renal ultrasound and cranial CT imaging) but with negative results. The inventors decided to focus their search for TSC associated deletions on the more proximal part of the candidate region, most of which is spanned by a Clal restriction fragment of approximately 340kb (Germino et al., 1992, Harris et al., 1990). Using pulsed field gel electrophoresis (PFGE) this fragment was assayed in 255 unrelated TSC patients with SM6, a single copy probe isolated from cosmid SMII (Figure 1) . The patients all fulfilled definitive diagnostic criteria as defined by Gomez (1988). Aberrant smaller fragments consistent with constitutional interstitial deletions were observed in 5 cases. As these changes were ' likely to involve the TSC gene, the inventors decided to characterise further the region containing the deletions. Mapping of PFGE deletions and genomic cloning Cosmid walking was initiated from the previously defined loci JCII and N54 (Germino et al. , 1990; Himmelbauer et al., 1991). The proximally directed walk established a series of overlapping clones spanning 200kb across the area of the TSC associated deletions, while the distally directed walk was hampered by a duplicated region homologous to sequences more proximal on 16p (Germino et al., 1992). A long range restriction map was constructed in genomic and cloned DNA which was consistent in size with that produced by Germino et al. (1992) although additional sites for Nrul and Mlul were identified. Mapping of SacII and BssHII sites enabled the unmethylated CpG islands to be located (Figure 2). The area was precisely mapped with EcoRI and other restriction enzymes and many fragments were subcloned (Figure 2 and Experimental Procedures for details). The sizes and positions of the five TSC deletions were more accurately determined by analysing Mlul and Nrul digested DNA. Successive hybridisations enabled fragments flanking or containing the deletion breakpoints to be identified. When suitable material was available a breakpoint fragment was identified in EcoRI, BamHI and/or Hindlll digests with probes immediately flanking the deletion, confirming the nature of the rearrangement. The precise position of each of the TSC deletions is summarised in Figure 2. Two deletions estimated at 32kb and 46kb, and two of at least 70kb were positioned distally and overlapped one another extensively. A fifth deletion of approximately 35kb was more proximally situated and was shown to be non-overlapping with at least 3 of the distal deletions (Figure 2). As each of these deletions was likely to involve part of the chromosome 16 TSC gene, a candidate gene that mapped into all of them was sought. Genes in the Region Harbouring Pulsed Field Deletions Subcloned probes and fragments from cosmids spanning the region of the TSC associated deletions were used to screen human foetal brain and human kidney cDNA libraries. The mapping of positive clones to the target area was confirmed by hybridisation to panels of somatic cell hybrids, containing derivative 16 chromosomes with breakpoints flanking this region; N- OH1, distal, and P-MWH2A, proximal (Figure 1), and a radiation hybrid Hyl45.19 which contains this area, as a positive control. Northern blot analysis using RNAs from various human cell lines indicated that the clones derived from four apparently unrelated genes. Hybridisation of the cDNA clones to digests of cosmid, genomic and hybrid DNA indicated the genomic distributions of the genes. Sequence analysis identified the polyA tail of each gene and established their transcriptional orientations. A gene, termed 0CTS2, with a transcript of 1.7kb (cDNA clones 0CTS2C and RCTS2) and a second gene termed 0CTS3 with a lkb transcript (cDNA clone 0CTS3C) mapped entirely within the four distal deletions, but did not extend as far as the proximal deletion in patient WS-53 (Figure 2). A 15kb transcript was recognised by two cDNA clones, 3A3 and AH4, and was termed 3A3. It mapped partly within the WS-53 deletion. Since the distal clone AH4 contained the polyA tail, the gene is transcribed from centromere to telomere and does not extend towards the distal deletions (Figure 2). The cDNA clones 2A6 and 4.9 detect an ~5.5kb transcript and were identified using an 18kb EcoRI fragment from cosmid ZDS5 (corresponding to the region subcloned in CW23 and CW21). A transcript of the same size was detected by CW26, a genomic probe which maps at a CpG island located within the four distal deletions (Figure 2). By means of a cDNA walk the 2A6 and 4.9 clones were connected to clones 4B2 and Al which mapped to the CW26 region confirming that this single gene is disrupted by all five PFGE deletions. This gene was therefore designated TSC2 and characterised in detail. TSC2 Expression Northern blot analysis indicates that TSC2 is widely expressed with the 5.5kb transcript seen in all cell lines tested, including those derived from brain, kidney, skin, liver, adrenal gland, colon and white blood cells. Expression has also been seen in all tissues tested, including liver, kidney and heart, and in lymphocytes, fibroblasts and biliary epithelium. The high level of TSC2 expression in fibroblasts made it possible to compare the level of transcription in fibroblasts derived from normal controls and TSC patients. In one family in which TSC has been shown to co-segregate with chromosome 16pl3.3 markers, but in which the mutation has not been identified, the affected members showed clearly reduced levels of TSC2 transcript. Transcripts from adjacent genes showed unaltered levels of expression. The combination of non-overlapping PFGE deletions affecting TSC2 and the reduced expression of the TSC2 transcript in TSC patients strongly suggests that the deletions inactivate the structural TSC determining gene rather than a regulatory element for a remote gene. To confirm that TSC2 is indeed a TSC determining gene we sought independent intragenic mutations. Intragenic Mutations Affecting TSC2 DNA samples from 260 unrelated TSC patients were screened for confirmatory rearrangements within TSC2 using cDNA sub-clones as hybridisation probes. All patients tested fulfilled the definitive diagnostic criteria of Gomez (1988) and included many of those previously studied by PFGE. In addition to those cases in which PFGE abnormalities had been found, aberrant bands were noted with multiple restriction enzymes in a further 5 patients. Southern analysis using a combination of genomic clones and small cDNA fragments as hybridisation probes demonstrated that these changes represented small deletions. The position of each deleted segment was confirmed relative to the genomic map of EcoRI, Hindlll and BamHI sites (Figure 5). The most 5' deletion found in patient WS-210, was not entirely intragenic as it also involved the 0CTS3 gene. The deletion spans 5-6kb and removes the genomic probe CW26 which contains TSC2 coding sequence. All four other deletions were shown to be entirely within TSC2. A deletion of approximately lkb in patient 5773 was shown to remove an intronic Hindlll site. In this case the mutation was also detected in the affected parent. In two further cases (WS-80 and 1737) deletions of approximately 3kb and 5kb respectively were identified. The parents of these cases were thought to be unaffected but were not available for analysis, making it impossible to confirm that the changes represented de novo mutations. In contrast, both clinically unaffected parents of patient WS-11 were available for analysis and the ~ 5kb deletion (which was not seen on PFGE) was shown to represent a de novo mutation. The deletion removes an intronic Hindlll site and the upstream intronic EcoRI site. The genomic probe CW18, which lies between these sites and detects the TSC2 transcript, was shown to be deleted. Leucocyte polyA RNA prepared from this patient showed an abnormal TSC2 transcript of " 4.5kb on Northern analysis. Together these findings confirm that TSC2 is the chromosome 16 tuberous sclerosis determining gene. Further deletions involving TSC2 Deletions involving both TSC2 and PKDl were identified and characterized in six patients in whom TSC was associated with infantile polycystic kidney disease. As well as the deletion in WS-53, those in WS-215 and WS- 250 also extended proximally well beyond the known distribution of PKDl and probably delete the entire gene. The deletion in WS-194 extended over the known extent of PKDl, but not much further proximally, while the proximal breakpoints in WS-219 and WS-227 lay within PKDl itself. Northern analysis of case WS-219 with probe JH8, which lies outside the deletion, showed a reduced level of the PKDl transcript but no evidence of an abnormally sized transcript (data not shown) . Analysis of samples from the clinically unaffected parents of patients WS-53, WS-215, WS-219, WS-227 and WS-250 showed the deletions in these patients to be de novo. The father of WS-194 was unavailable for study. In a further case (WS-212), renal ultrasound showed no cysts at four years of age but a deletion was identified which removed the entire TSC2 gene and deleted an Xbal site which is located 42 bp 5' to the polyadenylation signal of PKDl. Characterisation of TSC2 To further characterise the TSC2 gene, evolutionary conservation was studied and sequence analysis was performed. A 'zoo-blot' containing genomic DNA from various animal species revealed that the TSC2 gene was conserved throughout the higher vertebrates. Strong signals were obtained from primates and signals indicating lower homology were obtained from several other vertebrates, including rodents, marsupial and reptile. No signal was obtained from fish or non- vertebrate species. The TSC2 transcript was sequenced completely in both strands. All sequence was confirmed in at least two independent cDNA clones. The coding sequence obtained extends 5474bp (Figure 6). Despite repeated cDNA library rescreening, no clones extending further 5' could be identified. The available sequence approximates to the transcript size determined by Northern blot analysis. The cDNA contains an open reading frame (ORF) extending from nucleotide 1 through 5370. The second-best ORF is no more than 402bp. At nucleotide position 19 we found an in-frame start codon, matching the Kozak consensus (Kozak, 1987). At the 3 ' end we noted two partially overlapping polyadenylation signals (AATAAATAAA) at nucleotide 5425. The occurrence of this doublet may cause differential polyadenylation, since we found polyadenylation sites which differ by up to 15bp in four different cDNA clones. The total length of the predicted protein is 1784 amino acids with a calculated molecular mass of 198 Kd. There is no apparent signal peptide or signal peptidase cleavage site. Using the method described by Eisenberg et al. (1984) , we identified hydrophobic domains, four of which may represent membrane spanning regions. Within a predicted alpha-helical structure, a stretch of 22 amino acids, surrounded by a repeated motif of 9 amino acids, complied with the leucine zipper consensus (Landschulz et al., 1988) . A search for sequence homologies at protein level revealed a region of similarity between the predicted product of TSC2 and the GTPase activating protein GAP3 (or raplGAP) (Rubinfeld et al., 1991). The region extends over 58 amino acids and the level of residue identity fulfils the criteria of Sander and Schneider (1991) for structural homology. Of the first 39 amino acids, 14 are identical with murine GAP and with human GAP3. A core stretch of 17 residues contains identical or similar amino acids with only one mismatch (Figure 4) . Discussion The inventors have used a positional cloning strategy to identify a gene on chromosome 16 which is mutated in tuberous sclerosis. A number of questions concerning the biology of TSC and its relationship to other disorders can now be addressed. The TSC2 gene maps within the candidate region for the unidentified PKDl gene, causing autosomal dominant polycystic kidney disease type 1 (ADPKD1), as defined by Germino et al. (1992). As polycystic kidneys are a feature common to TSC and ADPKD1 (Bernstein and Robbins, 1991) the possibility of an aetiological link, as proposed by Kandt et al. (1992), must be considered. Renal cysts, however, have been reported in a chromosome 9-linked TSC family (Nellist et al. , 1993) and their presence is therefore not limited to chromosome 16-linked TSC. Furthermore, while TSC and ADPKD1 cysts are macroscopically similar, the epithelium lining TSC associated cysts is usually considered to be histologically distinct (Bernstein et al . , 1974) . Despite these observations it may be tempting to hypothesise that chromosome 16-linked forms of TSC and ADPKD1 are allelic variants. However, the inventors have not found any evidence that this is the case. The search for possible functional motifs in the sequence of the predicted protein, which the inventors have called tuberin, indicates several regions of interest. Four hydrophobic domains were identified which may be involved in membrane anchorage and four potential glycosylation sites were observed downstream of the last putative transmembrane domain. No sequence at the amino-terminus of the predicted protein matched the signal peptide structure as defined by von Heijne (1985) . However, the occurrence of several transmembrane domains without an apparent signal peptide was noted in the cystic f ibrosis-related protein CFTR (Riordan et al . , 1989). We also noted a periodic array of leucine residues (leucine zipper), a structure associated with protein-protein interaction. Experiments are in progress to determine the cellular localisation of tuberin, which will provide insight into the functional significance of the sequence motifs that have been identified. Because of the highly variable TSC phenotype, the genetic status of a patient's relatives may remain uncertain, even after extensive diagnostic investigation (Al-Gazali et al., 1989; Fryer et al. , 1990) . In this situation the identification of the causative mutation would be very helpful. Although a relatively small number of mutations are reported in this study, alternative approaches such as SSCP analysis (Orita et al., 1989) can be applied now that the TSC2 gene sequence is available. Identification of the TSC1 gene on chromosome 9 will also have to be achieved before the full mutational spectrum in TSC and the practicalities of DNA based diagnostics can be completely evaluated. The inventors have identified multiple deletional mutations affecting different parts of the TSC2 gene in unrelated TSC patients. This pattern, and the reduced expression of TSC2 seen in affected individuals, suggest that constitutional mutations in TSC are likely to be inactivating. The patchy focal nature of TSC associated lesions and the loss of heterozygosity which they exhibit (Green and Yates, 1993) suggest that reduction to the homozygous null state is required before cellular growth and differentiation become disordered. A similar combination of inactivating constitutional and somatic mutations have been clearly demonstrated in the Rb gene in retinoblastoma (Horowitz et al. , 1989) and more recently the NF1 gene in neurofibrosarcoma (Legius et al . , 1993); it has also been proposed in NF2 (Rouleau et al . , 1993) and VHL (Latif et al., 1993). It would seem likely, therefore, that TSC2 also behaves as a tumour suppressor gene as defined by Knudson's theory of carcinogenesis (Knudson, 1971). In view of these observations it is interesting that tuberin contains a region of homology to GAP3, which enhances the GTPase activity of p21rapl, a GTP binding protein thought to antagonise p21ras. The area of homology lies within a region known to be necessary for the catalytic activity of GAP3 (Rubinfeld et al., 1992) . It seems possible that tuberin may have GAP activity for p21rapl or another GAP protein involved in the control of cellular proliferation and differentiation. An analogous situation has already been demonstrated in NF1, where the normal regulation of p21ras is disrupted by mutations affecting the rasGAP homologue neurofibromin (Xu et al., 1990 and Martin et al., 1990). As the proteins involved in the various phakomatoses are identified, their functions and possible inter-relationships will be established. Experimental Procedures Pulsed Field electrophoresis High molecular weight DNA was isolated from peripheral blood in agarose plugs by standard methods (Hermann et al., 1987) and digested according to the manufacturers recommendations. Blocks were loaded into the wells of 1% agarose gels and electrophoresis carried out using a BioRad CHEF DR II or a similar apparatus and programs appropriate to the varying resolutions required. Southern Blot Analysis Genomic DNA was extracted from peripheral blood by standard methods. 5-8μg DNA was digested with restriction enzymes, electrophoresed through agarose gels and blotted to nylon filters as described (Sambrook et al. , 1989). Probes were labelled by the random-primer method (Feinberg and Vogelstein, 1984). For probes containing repetitive elements, lOng of labelled DNA was pre-associated with 0.1-lmg denatured sonicated total human DNA in a total volume of 200μl at 650C for 1-5 hr. prior to hybridisation. If required filters were additionally prehybridized with lOOμg/ml denatured sonicated total human DNA and salmon sperm DNA. Filters were hybridised, washed as described (Sambrook et al. , 1989) and exposed to autoradiographic film with an intensifying screen at -70°C. DNA Probes and Somatic Cell Hybrids Some of the probes used in this study have been described previously: MS205.2 (D16S309; Royle et al., 1992); GGG1 (D16S259; Germino et al., 1990); 16AC2.5 (D16S291; Thompson et al., 1992) and N54 (D16S139; Himmelbauer et al., 1991). A number of new probes were also isolated during the course of this study: SM6, a 2.3kb Sau3A fragment from SMII; BFS2, a l.δkb BssHII fragment of CC1-2; SM9, a 7kb EcoRI fragment of CBFSl; CW9 a lkb EcoRI/NotI segment of CBFSl; CW15 a lOkb EcoRI/NotI fragment of CW9D; CW24 and CW26 are 0.9kb and 0.4kb, SacII and SacII/SacI fragments, respectively, of CW9D; CW13 and CW12 are EcoRI/NotI fragments of 2.2kb and 2.0kb, respectively, from CW9D; CW18, CW20 are EcoRI/NotI fragments of 3kb and 16kb respectively from CW12I, JHl, a 4.4kb BamHI fragment of CW12I; and CW23 and CW21 are 14kb and 3.5kb Notl/EcoRI fragments, respectively, of JHIK. All new probes except SM6, BFS2, CW26 and CW21 contain repetitive sequences and were hybridised in the presence of denatured, sonicated human DNA (75ug/ml) and washed in 0.05 x SSC, 0.2% SDS at 650C. The somatic cell hybrid N-OHl and the radiation hybrid, Hyl45.19 have been described previously (Germino et al, 1990; Himmelbauer et al, 1991). The P- MWH2A hybrid contains the derivative chromosome 16qter - 16pl3.3 : : 7q32 - 7qter and was isolated from a subject, MW, who has a balanced translocation. P-MWH2A was produced by fusing lymphoblastoid cells from MW with APRT deficient mouse erythroleukemia cells by the method of Deisseroth and Hendrick (1979) . The breakpoint in this hybrid has been localised to the region between 16AC2.5 and the adjacent Clal site (see Figure 1 ) . RNA Isolation and Northern Blot Analysis RNA was extracted from cell-lines and tissues by the acid phenol method of Chomczynski and Sacchi (1987) . mRNA was isolated from total RNA using a biotinylated oligo (dT) primer and streptavidin coupled paramagnetic particles (PolyATtract mRNA Isolation System, Promega) . RNA was separated in denaturing formaldehyde gels and Northern blotted by standard procedures. Hybridisation and washing of Northern blots was as described for Southerns. Cosmid Walking Cosmids were obtained from several different libraries: Los Alamos Chromosome 16 specific library (Stallings, et al. 1990) and total genomic cosmid libraries 412 and IG328 (Integrated Genetics) and 961200 (Stratagene) . Successive cosmid walks were made by mapping each cosmid, isolating end clones and rehybridising the libraries using conditions to repress repetitive sequences if necessary. A cosmid/genomic EcoRI map was produced and the location of cosmids was checked by mapping on hybrid panels, PFGE and fluorescence in situ hybridisation. cDNA Isolation and Characterisation Screening for cDNAs was performed using standard phage plating, filter lift and clone purification techniques in commercial libraries derived from human fetal brain (Clonetech, Stratagene) and human adult kidney (Clonetech). Filters were lifted as described by Sambrook (Sambrook, 1989). Repetitive sequences were suppressed as described above. After overnight hybridisation at 650C, filters were washed as described (Sambrook, 1989). All positive clones were subcloned into one of the pBluescript or pUC vectors and sequenced with a Pharmacia A.L.F. or ABI model 373A automated sequencer according to the manufacturers protocol, or manually. References Al-Gazali, L.I., Arthur, R.J., Lamb, J.T., Hammer, H.M., Coker, T.P., Hirschmann, P.N., Gibbs, J., and Mueller, R.F. (1989). 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Claims: CLAIMS 1. An isolated, purified or recombinant nucleic acid sequence comprising:- (a) a TSC2 gene or its complementary strand, (b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above, (c) a fragment of a molecule defined in (a) or (b) above. 2. A sequence according to claim 1 comprising a purified DNA molecule having a sequence corresponding to all or a portion of the nucleotide sequence of Figure 3, or a complementary sequence, or a sequence which hybridises to any of the above sequences. 3. A sequence according to claim 1 or claim 2 comprising a purified DNA molecule characterised as follows:- (i) it is present in the telomeric chromosomal band 16p 13.3, ( ii ) it is mutated in TSC patients, (iii) it lies between markers GGG1 and 16AC2.5. 4. A purified DNA molecule according to Claim 3, wherein the sequence is contained in cosmids ZDS-5 and LADS-4. 5. A nucleic acid sequence comprising a mutant TSC2 gene, selected from those wherein (a) [WS-13] 32kb are deleted flanked by CW13 andCW9; (b) [WS-9] about 46kb are deleted with breakpoints in SM9 and CW12; (c) [WS-211] about 75kb are deleted with breakpoints between CW9 and CW15 distally, and between CW23 and CW21 proximally; (d) [WS-97] about 75kb are deleted between BFS2 and SM9 distally, and within CW20 proximally; (e) [WS-53] a large deletion between, distally, CW23 and JHl such that about 0.6kb of TSC2 is deleted; (f) [WS212] about 75kb are deleted between SM9-CW9 distally and the TSC2 3'UTR proximally as shown in Figure 8; (g) [WS-215] about 160kb are deleted between CW20 and CW10-CW36 as shown in Figure 8; (h) [WS-227] about 50kb are deleted between CW20 and JH11 as shown in Figure 8; (i) [WS-219] about 27kb are deleted between JHl and JH6 as shown in Figure 8; (j) [WS-250] about 160kb are deleted between CW20 and BLu24 as shown in Figure 8; and (k) [WS-94] about 65kb are deleted between CW20 and CW10. 6. An RNA molecule comprising an RNA sequence corresponding to a DNA sequence according to any of claims 1 to 5. 7. A nucleic acid probe having a sequence according to any of the preceding claims and optionally including a label. 8. A nucleic acid sequence according to any preceding claim, wherein the nucleic acid sequence encoding TSC2 is operably linked to transcriptional and/or translational expression signals. 9. An isolated, purified or recombinant polypeptide comprising a TSC2 protein or a mutant or variant thereof or encoded by a sequence according to any of claims 1 to 8 or a variant thereof having substantially the same activity as the TSC2 protein. 10. A polypeptide according to claim 9, wherein the TSC2 protein has the amino acid sequence according to the amino acid sequence of Figure 3. 11. An anti-TSC2 antibody or a labelled anti-TSC2 antibody. 12. A method for screening a subject to determine whether said subject is a TSC2-associated disorder carrier or a patient having a TSC2-associated disorder, which method comprises detecting the presence of and/or evaluating the characteristics of TSC2 DNA, TSC2 RNA and/or TSC2 polypeptide in a biological sample from said patient. 13. A method according to claim 12 which comprises detecting and/or evaluating whether the TSC2 DNA is . mutated, deleted, aberrant or otherwise abnormal, or is not expressing normal TSC2 protein. 14. A method according to claim 12 or 13, wherein the detection and/or evaluation includes the step of comparing the results thereof with results obtained using a mutated sequence according to claim 5. 15. A method according to any of claims 12 to 14, wherein said screening includes applying a nucleic acid amplification process to said sample to amplify a fragment of the TSC2 DNA or cDNA corresponding to the TSC2 RNA. 16. A vector comprising the nucleic acid sequence of any of claims 1 to 8. 17. A host cell transfected or transformed with a vector according to claim 16. 18. The use of a vector according to claim 17 or a nucleic acid sequence according to any of claims 1 to 10 in gene therapy and/or in the preparation of an agent for treating or preventing a TSC2-associated disorder. 19. A method of treating or preventing a TSC2- associated disorder which method comprises providing to a patient in need thereof a functional TSC2 gene to affected cells in a manner that permits expression of TSC2 protein (tuberin) therein and/or a transcript produced from a mutated chromosome which is capable of expressing functional TSC2 protein therein and/or by administering to the patient TSC2 protein or a tuberin functional mimic. 20. A method for suppressing tumour development and/or preventing unrestrained cell-division in a patient with or without TSC, which method comprises providing to the patient a functional TSC2 gene in a manner that permits expression of TSC2 protein (tuberin) therein and/or a transcript produced from a mutated chromosome which is capable of expressing functional TSC2 protein therein and/or by administering to the patient TSC2 protein or a tuberin functional mimic. 21. A diagnostic kit for carrying out a method according to any of claims 13 to 19, comprising nucleic acid primers for amplifying a fragment of a sequence according to any of Claims 1 to 10. 22. A diagnostic kit for carrying out a method for determining whether said subject is a TSCl-associated disorder carrier or a patient having a TSC2-associated disorder, which includes a nucleic acid probe capable of hybridising to a sequence according to any of claims 1 to 10. 23. A method of producing a protein or polypeptide having the amino acid sequence of Figure 3, which comprises culturing a host cell transfected with the vector of Claim 16 under conditions favourable for expressing said protein or polypeptide and thereafter isolating said protein or polypeptide.

Inventors: BREUNING MARTIN HENDRIK, BROOK-CARTER PHILLIP, HALLEY DIRKJE JORIJNTJE JOHANN, HARRIS PETER CHARLES, HESSELING ARJENNE LIQUE WILHEL, JANSSEN LAMBERTUS ANTONIUS JAC, MAHESHWAR MAGITHA, NELLIST MARK DAVID, ROELFSEMA JEROEN HENDRIK, SAMPSON JULIAN, VAN DEN OUWELAND ANNA MARIA WI, WARD CHRISTOPHER JAMES, BREUNING, MARTIN, HENDRIK, BROOK-CARTER, PHILLIP, HALLEY, DIRKJE, JORIJNTJE, JOHANNA, HARRIS, PETER, CHARLES, HESSELING, ARJENNE, LIQUE, WILHELMA, JANSSEN, LAMBERTUS, ANTONIUS, JACOBUS, MAHESHWAR, MAGITHA, NELLIST, MARK, DAVID, ROELFSEMA, JEROEN, HENDRIK, SAMPSON, JULIAN, VAN DEN OUWELAND, ANNA, MARIA, WILHELMINA, WARD, CHRISTOPHER, JAMES
Applicants: BREUNING MARTIN HENDRIK, BROOK CARTER PHILLIP, DEN OUWELAND ANNA MARIA WILHEL, HALLEY DIRKJE JORIJNTJE JOHANN, HARRIS PETER CHARLES, HESSELING ARJENNE LIQUE WILHEL, JANSSEN LAMBERTUS ANTONIUS JAC, MAHESHWAR MAGITHA, MEDICAL RES COUNCIL, NELLIST MARK DAVID, ROELFSEMA JEROEN HENDRIK, SAMPSON JULIAN, UNIV ERASMUS, UNIV LEIDEN, UNIV WALES MEDICINE, WARD CHRISTOPHER JAMES, BREUNING, MARTIN, HENDRIK, BROOK-CARTER, PHILLIP, ERASMUS UNIVERSITY ROTTERDAM, HALLEY, DIRKJE, JORIJNTJE, JOHANNA, HARRIS, PETER, CHARLES, HESSELING, ARJENNE, LIQUE, WILHELMA, JANSSEN, LAMBERTUS, ANTONIUS, JACOBUS, LEIDEN UNIVERSITY, MAHESHWAR, MAGITHA, MEDICAL RESEARCH COUNCIL, NELLIST, MARK, DAVID, ROELFSEMA, JEROEN, HENDRIK, SAMPSON, JULIAN, UNIVERSITY OF WALES COLLEGE OF MEDICINE, VAN DEN OUWELAND, ANNA, MARIA, WILHELMINA, WARD, CHRISTOPHER, JAMES
Application year: 1994
Main classification: C12N15
IPC codes: A61K31, A61K38, A61K39, A61K48, A61P13, A61P15, A61P35, C07H21, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, C12R1, G01N33